The present invention relates in general to cell and tissue transplantation techniques. More particularly, the present invention is directed to techniques for transplanting populations of retinal pigment epithelium (RPE) cells as a monolayer to the subretinal region of the eye, and to methods for preparing implants comprising monolayers of RPE cells for transplantation.
The retina is the sensory epithelial surface that lines the posterior aspect of the eye, receives the image formed by the lens, transduces this image into neural impulses and conveys this information to the brain by the optic nerve. The retina comprises a number of layers, namely, the ganglion cell layer, inner plexiform layer, inner nuclear layer, outer plexiform layer, outer nuclear layer, photoreceptor inner segments and outer segments. The outer nuclear layer comprises the cell bodies of the photoreceptor cells with the inner and outer segments being extensions of the cell bodies. The choroid is a vascular membrane containing large branched pigment cells that lies between the retina and the sclerotic coat of the vertebrate eye. Atop the choroid is a membrane 1-5 microns in thickness essentially composed of collagen, known as Bruch's membrane.
Immediately between Bruch's membrane and the retina is the retinal pigment epithelium which forms an intimate structural and functional relationship with the photoreceptor cells. Among the functions performed by RPE cells is the phagocytosis of outer segment debris produced by the photoreceptors. It is believed that failure of the RPE cells to properly perform their functions such as digestion of outer segment debris leads to the eventual degeneration and loss of photoreceptor cells.
In the leading causes of visual impairment in western industrialized countries, such as age-related macular degeneration (AMD), both photoreceptors and the underlying RPE are compromised, or have degenerated. A further aspect of AMD is the frequent appearance of subretinal neovascular membranes which grow through the Bruch's membrane and the RPE and tend to hemorrhage and leak fluids into the subretinal space.
In an effort to recover what was previously thought to be an irreparably injured retina, researchers have suggested various forms of grafts and transplantation techniques, none of which constitute an effective manner for reconstructing a dystrophic retina. The transplantation of retinal cells to the eye can be traced to a report by Royo et al., Growth 23: 313-336 (1959) in which embryonic retina was transplanted to the anterior chamber of the maternal eye. A variety of cells were reported to survive, including photoreceptors. Subsequently del Cerro was able to repeat and extend these experiments (del Cerro et al., Invest. Ophthalmol. Vis. Sci. 26: 1182-1185, 1985). Soon afterward Turner, et al. Dev. Brain Res. 26:91-104 (1986) showed that neonatal retinal tissue could be transplanted into retinal wounds.
Li and Turner, Exp. Eye Res. 47:911 (1988) have proposed the transplantation of retinal pigment epithelium (RPE) into the subretinal space as a therapeutic approach in the RCS dystrophic rat to replace defective mutant RPE cells with their healthy wild-type counterparts. According to their approach, RPE were isolated from 6- to 8-day old black eyed rats and grafted into the subretinal space by using a lesion paradigm which penetrates through the sclera and choroid. A 1 .mu.l bolus injection of RPE (40,000-60,000 cells) was made at the incision site into the subretinal space by means of a 10 .mu.l syringe to which was attached a 30 gauge needle. However, while this technique is marginally appropriate for immature RPE cells, with mature cells it leads to activation and transformation of these cells which damages eye and retinal tissue.
Lopez et al., Invest. Ophthalmol. Vis. Sci. 30: 586-589, 1989, also reported a procedure for the transplantation of dissociated RPE cells. In this procedure, RPE cells were obtained from normal, congenic, pigmented rat eyes by trypsin digestion. These freshly harvested, dissociated RPE cells were injected into the subretinal area of the eyes of dystrophic RCS rats via an incision through the sclera, choroid and neural retina. Comparable to the Li and Turner approach discussed above, this procedure destroys the organized native structure of the transplanted RPE cells, which take the form of a confluent monolayer in a healthy eye. Moreover, the procedure is of questionable value for the transplantation of mature RPE cells. When mature RPE cells are transplanted in dissociated form, experimental results indicate that they are likely to become activated, migrate into the subretinal space, and as noted by Lane, C., et al., Eye (1989) 3, 27-32, invade the retina and vitreous. This activation of the transplanted, mature RPE cells can result in such pathologies as retinal pucker, massive subretinal fibrosis, retinal rosette formation, retinal detachment, and proliferative vitreoretinopathy.
The difficulties discussed above associated with the transplantation of mature RPE cells is problematic for human transplantation since available supplies of immature human RPE donor tissue are extremely limited. Moreover, the inability to use mature cells effectively prevents transplantation using autologous RPE tissue, which otherwise would be desirable to avoid the complications involving potential immunological responses faced by non-autologous transplants. Since the victims of AMD are predominantly older adults, in most cases utilizing autologous tissues for transplants would necessarily entail the use of mature human RPE cells.
It is believed by the present inventor that it is necessary to maintain adult human RPE cells substantially as a monolayer to achieve their successful transplantation and to avoid the problems associated with activation of RPE cells. Although not wishing to be limited to a particular theory, it is thought that the cell-to-cell contact inhibition provided by an intact monolayer, supplemented by adherence to a substrate, mitigates against RPE cell activation. Moreover, a monolayer structure for the RPE provides a proper foundation for the maintenance of the photoreceptor cells in an organized outer nuclear layer structure and for proper growth and arrangement of inner and outer segments, believed by this inventor to be advantagous to restore a reasonable degree of vision. The requirement that the photoreceptors be maintained in an organized structure is based on the well known optical characteristics of photoreceptors (outer segments act as light guides) and clinical evidence showing that folds or similar, even minor, disruptions in the retinal geometry can severely degrade visual acuity.
Additionally, in cases of AMD where subretinal neovascular membranes have appeared, prior to RPE transplantation, such membranes will need to be removed to prevent subretinal edemas and hemorrhaging of these membranes. In practice, removal of the neovascular membrane results in removal of the native RPE and Bruch's membrane as well.
A critical impediment to the transplantation of RPE cells as a monolayer is the fragility of the intercellular structure of RPE relative to the rigors of manipulation during transplantation to the subretinal area. Moreover, providing satisfactory support to the RPE cells during this process is complicated by the fact that the support must either be removed subsequent to transplantation, to avoid compromising metabolic exchange between the choroid and the overlying retina, or be compatible with such ongoing physiological activity. Thus, a method is needed wherein an implant comprising a monolayer of RPE cells is prepared and transplanted in which the component supporting the monolayer of RPE cells, upon transplantation to the subretinal area and exposure to a set of predetermined conditions, does not impede normal eye tissue function. Further, a Bruch's-like membrane for attachment of the transplanted RPE cells will be needed in those cases where neovascularization has occurred and the native Bruch's membrane is removed.